Quantification of mRNA using real-time RT-PCR


The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a “gold” standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.

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Figure 1: Steps involved in planning a RT-qPCR assay.
Figure 2: RT-qPCR experimental workflow.
Figure 3: The 3′:5′ assay used to estimate mRNA integrity.
Figure 4: Optimization of primer concentration.
Figure 5: Acceptable and unacceptable standard curves.
Figure 6: Outline of reverse transcription protocol.
Figure 7: The relationship between amplification plot and standard curve.
Figure 8: Invalid Ct generated by spurious amplification plot.
Figure 9: Amplification plots demonstrating the importance of appropriately adjusting the baseline.
Figure 10: SYBR Green melt curve demonstrating the co-amplification of an mRNA target (lower Tm) and genomic DNA with a short intron.
Figure 11: SYBR Green melt curve demonstrating the appearance of primer dimers in a poorly designed assay.
Figure 12: Comparison of SYBR Green I and TaqMan amplification plots.


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We acknowledge numerous fruitful discussions with J. Huggett, M. Kubista, R. Mueller, S. Mueller, M. Pfaffl, G. Shipley and J. Vandesompele. We are grateful to N. Gerke (Eppendorf AG) and J. Stolte (EMBL Heidelberg) for the experiment described in Figure 4, which was conducted at the 3rd EMBO RT-qPCR workshop at EMBL Heidelberg, 2006.

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Correspondence to Stephen A Bustin.

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Nolan, T., Hands, R. & Bustin, S. Quantification of mRNA using real-time RT-PCR. Nat Protoc 1, 1559–1582 (2006). https://doi.org/10.1038/nprot.2006.236

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